重组腺病毒p53 上调凋亡调控因子通过凋亡通路增强胶质瘤细胞对替莫唑胺敏感性的体外研究

目的 探讨转染p53 上调凋亡调控因子(PUMA)的人脑胶质瘤细胞对替莫唑胺敏感性增强的作用机制.方法 将人脑胶质瘤细胞U87MG 分为正常对照组、空载体组和实验组进行培养,分别将感染复数(MOI) ＝50 的空载体腺病毒(Ad-△ BH3)和携带PUMA 的重组腺病毒(Ad-PUMA)转染U87MG 细胞,转染后用MTT 比色法测定各组细胞的存活率并计算替莫唑胺(TMZ)IC50,流式细胞仪检测细胞周期的变化,应用TUNEL 法检测细胞的凋亡情况.Western blot 检测凋亡相关蛋白p53、Bax 的表达.结果 外源性PUMA 基因在Ad-PUMA 转染U87MG 48 h 后,随着时间延长,PUMA 表达使U87MG 细胞的增殖能力降低并诱导凋亡,转染24 h、48 h、72 h 的生长抑制率分别为17.3%、35.6%、43.3%,凋亡率分别为20.3%、31.4%、45.4%.正常对照组、Ad-PUMA、Ad-△BH3 组细胞的TMZ IC50分别为(15.0 ±1.9)μmol/L、(2.3 ±0.1)μmol/L 和(14.4 ±1.6)μmol/L.PUMA 表达的U87MG 细胞DNA 合成受到抑制,周期阻滞在G2 期;Western blot 检测显示p53 表达在各组中差异无统计学意义(P ＞0.05)、PUMA 和Bax 在实验组中表达较对照组强,差异有统计学意义(P ＜0.01).结论 Ad-PU-MA 转染可有效增强人脑胶质瘤细胞U87MG 对TMZ 的敏感性,抑制人脑胶质瘤细胞U87MG 的增殖,通过诱导细胞G2 期阻滞促进其凋亡,促凋亡机制不依赖于p53.

Studies on effect of recombinant adenovirus PUMA on sensitivity of human glioblastoma cells to temozolomide through enhanced apoptosis pathways in vitro

Objective To investigate the effect of PUMA on the sensitivity of human glioblastoma U87MG cells to temozolomide and its related mechanisms.Methods U87MG cells under culture were divided into the normal control group,mock group and experiment group.After 24 hours of culture,the mock and experiment group were transfected with mock vector（Ad-△BH3）and the recombinant adenovirus carrying PUMA（Ad-PUMA）respectively at a multiplication of infection（MOI）of 50.The proliferation activity of cells and IC50 were detected by MTT assay,the apoptosis effect and the change of cell cycle determined by Hoechst stain and flow cytometry（FCM）technology respectively.The expression of related proteins was revealed by the method of Western blot.Results Exotic PUMA gene was expressed in U87MG cells transfected with Ad-PUMA,which could inhibit the proliferation of U87MG cells.The inhibition rate of proliferation 24,48,72 h after transfection was 17.3%,35.6%,43.3% and apoptosis rate was 20.3%,31.4%,45.4% respectively.Results：The TMZ IC50 values of PBS group,Ad-PUMA and Ad-△BH3 group cells were（15±1.9）,（2.3±0.14）and（14.4±1.6）μmol/L respectively,with the sensitivity to the TMZ of Ad-PUMA group cells increased by 7.0 folds.PUMA overexpressing U87MG cells showed the DNA synthesis was inhibited and arrested in G2 phrase.The results of Western blot showed that after 72 h of transfection the PUMA and Bax protein showed increased expression（P0.01）.There was no significant difference in the expression of p53 among these groups（P0.05）.Conclusions PUMA gene transfection greatly enhances the sensitivity of U87MG cells to TMZ-induced apoptosis and can effectively inhibit the proliferation and promote the apoptosis of U87MG cells.The mechanism of apoptosis mainly relates to induce cell cycle G2 arrest and apoptosis in vitro.